Typically, organic solvent extraction and/or precipitation is utilized to remove various proteins and peptides from sample matrices before liquid chromatography analyses are performed on biological samples of, for example, blood serum, urine, body cells and brain tissue. Such conventional procedures may take between one and six hours and involve tedious and complex steps, including vortexing, centrifuging, drying, and possible reconstitution and repeated drying of the sample material. Because of the numerous steps and extensive handling of the sample material required in such typical processes, contamination of the sample during such handling is not unusual. Consequently, loss of samples poses a serious problem and can result in a low percentage recovery of analytes and a generation of less reproducible test data.
The above-discussed problems can present serious difficulties in validating the results obtained from biological sample analysis. Reproducibility of data is critical for many preclinical and clinical pharmacokinetic studies, hence simplicity that reduces data error or loss is highly desirable both in the apparatus and in the method for handling samples.
Numerous systems and methods for performing liquid chromatography analyses are known. They tend to involve large numbers of cooperating elements and the performance of correspondingly numerous procedural steps.
U.S. Pat. No. 4,751,185 to Ono et al., discloses a method for hop bittering components which comprises a step of separating and analyzing hop bittering components by liquid chromatography. In the disclosed apparatus, a flow line for liquid chromatography and a flow line for pretreatment of a sample are linked by a high pressure switching valve. The polar mobile phase from a supply is introduced into a precolumn which includes an auto injector and is then transferred to a separation column as the flow line is switched over by operation of a high pressure switching valve.
U.S. Pat. No. 4,913,821 to Melcher et al., discloses a method for the determination of phenols in water by adding a halogenating agent to the water, e.g., by employing a bromate-bromide reaction to generate tribromide ions which react with the phenols to form bromo-derivatives of the phenols. The reactive phenol is permeated across a silicone rubber membrane into a liquid extractant such as a solution of sodium hydroxide and acetonitrile. This extractant removes a halogenated derivative of the phenol. The haloderivative is determined by diverse phase liquid chromatography. The sample and reagent pass to a membrane cell and to an injection valve from a reservoir. By selective positioning of valves in the system, the extractant is passed to the membrane or purged to waste.
U.S. Pat. No. 4,952,514 to Haddad, discloses a method for the analysis of a metal selected from precious metals such as gold, palladium and platinum when these may be present in solution as a cyanide complex, using ion-interaction reverse phase liquid chromatography. In a first washing step, a first eluent is used to coat the surface of a concentrator column with an ion-interaction reagent. This is achieved through the operation of pneumatically actuated low pressure valves, a pump and six-port high pressure switching valves. The valves are all combined and operated in a single automated switching unit.
Other examples of relevant analytical apparatus and methods include U.S. Pat. Nos. 4,577,492 (to Holba et al.), 4,454,043 (to Ting et al.), 3,923,460 (to Parrott et al.), and 4,446,105 (to Dinsmore et al.). Such prior art indicates that the use of a switching valve and one or more other types of valves, including injection valves, is known generally in the art of liquid chromatography.
There is, however, a need for simplified apparatus and a method employing known switching valves, pumps, injection valves and control mechanisms for expeditious and low cost preparation of biological samples for liquid chromatography analyses. This need, as explained more fully hereinbelow, is met by the apparatus and method of the present invention which employs direct injection of biological samples with the use of a switching valve operable in an extremely simple manner to enable quick and low cost liquid chromatography analysis of biological tissue samples.